I. Field of the Invention
The present invention relates generally to the fields of immunology, microbiology, infectious diseases and medicine. In a more particular embodiment, it concerns methods and compositions including an exotoxin protein, such as α-hemolysin, for producing an immune response to a bacterium.
II. Background
The current methods for treating S. aureus pneumonia rely on antimicrobial drugs against which the organism has a remarkable propensity to acquire resistance. The pathogenesis of staphylococcal infections relies on many different virulence factors such as secreted exotoxins. Previous studies have shown that deletion of single genes encoding such factors causes either no defect or results in only modest reduction of virulence. However, studies of S. aureus pneumonia in a murine model system conducted by the inventors unexpectedly defined α-hemolysin, also known as alpha toxin, as a critical virulence factor in the pathogenesis of the disease, as a mutant strain lacking this exotoxin was avirulent. Alpha-hemolysin is a member of a family of bacterial cytotoxins that is secreted by S. aureus and is capable of inserting into the cell membrane of a multitude of eukaryotic cells. The protein is secreted as a monomer, however it assembles into a heptameric ring structure on the surface of eukaryotic cells. The assembled toxin inserts into the host cell membrane, forming a pore that contributes to cellular injury and death by disrupting the integrity of the membrane. Several biochemical studies have defined the amino acid residues within the α-hemolysin monomer that facilitate binding to the host cell, heptamer formation and host cell lysis.
The development of staphylococcal vaccines is hindered by the multifaceted nature of staphylococcal invasion strategies. It is well established that live attenuated microorganisms are highly effective vaccines, presenting a number of antigens to the subject's immune system. Immune responses elicited by such vaccines are often of greater magnitude and of longer duration than those produced by non-replicating or multi-component immunogens. One explanation for this may be that live attenuated strains establish limited infections in the host and mimic the early stages of natural infection as well as presenting a number of antigens to the immune system.
A number of references describe the inclusion of a α-hemolysin (Hla) component in a vaccine, some of which describe a chemically or heat attenuated Hla toxoid. See U.S. Pat. No. 4,327,082 for example. Other references have described immunizing a human with a multi-component toxoid vaccine and isolating Hla neutralizing antibodies for use in passive immunization. See U.S. Pat. No. 4,027,010. Adlam et al., (1977) have tested the effectiveness of purified Hla to protect against mammary infections. Adlam et al. observed a reduction in the “blue breast form” of mastitis, but did not see protection against the local chronic abscess form of staphylococcal disease. Adlam et al., attribute this observation to the insufficiency of Hla alone to protect against a multi-factorial disease state such as the local chronic abscess form of staphylococcal infection.
Bhakdi et al. (1994) have described the reduced toxicity of Hla having a mutation at residue 35 and describe administration of such a mutant to a rabbit without killing the rabbit. Menzies and Kernodle (1996) describe a similar H35L mutant of Hla and its use to produce antibodies in rabbits that can later be purified and used in passive immunity experiments. Menzies and Kernodle also describe the difficulty and expected failure of producing protection using a single component vaccine; they state “The great diversity of S aureus as a pathogen and the multitude of virulence factors which it produces make it unlikely that a single immunologic target such as alpha toxin would be effective as a vaccine candidate.” The inventors note that none of these references address the effectiveness of any composition to protect against or treat staphylococcal pneumonia.
The state of the art is such that one of skill in the art would not consider a recombinant Hla alone or substantially alone as an effective antigen for protecting against staphylococcus infection, particularly respiratory infections of staphylococcus or the indirect effects of staphylococcal respiratory infection. Thus, those of skill in the art would have no expectation of Hla, administered as a primary vaccine component in the absence or substantial absence of other Staphylococcal antigen(s), evoking an immune response sufficient for protecting a subject from or treating a subject with respiratory infection or staphylococcal associated pneumonia.
There remains a need in the art for additional compositions and methods for preventing and/or treating staphylococcal infection of the lungs, as well as the attenuation or amelioration of the secondary effects of such an infection.